The present invention relates, in general, to the efficient separation of molecules such as DNA and proteins, and more particularly to a separation device including nanofluidic channels of different sizes for providing alternate thin and thick regions along a channel to act as a filtering or sieving structure.
The separation of molecules according to their sizes is an essential step in biology and other fields and in analytical procedures such as chromatography, DNA sequencing or genome mapping. Conventional methods for separating molecules include electrophoresis and chromatography, which utilize the different transport properties (mobility) of different molecules in a solution-filled capillary or column. In many cases, additional sieving material, such as a gel matrix, is required to obtain sufficient separation of the molecules to permit analysis. In a conventional gel electrophoresis, as an example, molecules such as DNA molecules are separated during an electric-field-driven motion in a highly restrictive gel matrix, because the mobility of the molecules is dependent on their length. However, this length-dependence of molecule mobility vanishes for DNA molecules longer than about 40,000 base pairs, mainly because the molecules tend to be more stretched and oriented in the direction of the applied electric field. Molecules as long as 10,000,000 base pairs can be separated by pulsing the electric field (pulsed field gel electrophoresis), but this process is usually very time consuming and inefficient.
To obtain better efficiency and control for separation process, the use of an artificial system using a precisely defined microchannel structure as a molecular sieve has been suggested. However, initial attempts to produce efficient artificial gel systems were hindered by poor understanding of the molecular dynamics in the microchannels. It has been found that the conformation (shape) of DNA or other polymer molecules has a direct impact on their motion in a restrictive medium because the interaction cross section of the molecules with obstacles is changed with conformational change. In free solution, polymer molecules such as DNA have a spherical shape in their equilibrium state, and the size of this equilibrium shape is characterized by a radius of gyration (Ro) of the molecule. In the separation process of DNA or other polymers, it is important to maintain the conformation of the molecule in its equilibrium shape as much as possible, because otherwise the polymeric molecule will stretch out in the direction of the motion, rendering the mobility of the molecule length (size) independent. This is because there is minimal difference in their interaction with a retarding matrix such as gel or obstacles.
In terms of the fabrication of artificial gel systems, current photolithography techniques are limited in resolution at about the 1 micrometer level. Therefore, one cannot easily make constrictions or obstacles small enough for the separation of important molecules (DNA, proteins etc). Electron beam lithography can fabricate smaller features but it generally is too expensive, and it is difficult to produce a large-area device with this process.
It became clear that a more careful design of a separation device, combined with an inexpensive technique that can produce many ultrasmall constrictions over a large area, is essential in developing a functioning molecular separation device.
When molecules become relaxed or are in their equilibrium spherical shape, their interaction with a retarding matrix can be dependent on the molecule""s radius of gyration (Ro), and in turn on the length of the molecule. Accordingly, a design for a molecule sieving structure should include a somewhat open area where molecules can relax, as well as narrow constrictions that can serve as a molecular sieve.
It is, therefore, an object of the present invention to provide a separation device incorporating nanofluidic constrictions (thin regions) and obstacle free regions (thick regions), through which molecules can be caused to flow either by electrophoresis or by non-electric forces.
Briefly, the device of the invention provides a flow channel incorporating alternating thin and thick regions which operate as a filter, or sieving structure. The thin regions are sufficiently small to act as constrictions to the flow of small objects, such as DNA molecules, proteins, cells, viruses, or other similarly-sized particles, while the thick regions allow molecules to relax for more efficient separation at the thin region. To this end, the thick region depth may be made comparable to, or substantially larger than, the size of a molecule (for example, the radius of gyration Ro for polymer molecules) to be sieved. Also the thin region depth may be made substantially smaller than the size of the molecule or other object to be sieved. Although the device of the invention can be used to filter a variety of objects, the following description will be in terms of molecules, and particularly DNA molecules for convenience.
Accordingly, the present invention is directed to a nanofluidic channel in which the motion of molecules such as DNA molecules is characterized by the provision of molecular traps. In accordance with the invention, an elongated nanofluidic channel is provided with alternating regions of thick and thin gaps along its length. The equilibrium spherical shape of a molecule such as DNA or protein has a radius of gyration Ro, which is the shape the molecule assumes when it is relaxed in an open region, such as in the thick regions of the channel. If the molecule is forced to enter a constriction that is much less than Ro, the molecule has to be deformed from its equilibrium shape. Since such a deformation is entropically unfavorable, a driving force is required to force the molecule to enter the constriction. This effect is referred to as the entropic trapping of a long polymer, and this effect is crucial in the operation of present invention.
The entropic trapping effect can be utilized in operations such as molecular trapping, molecular band formation, molecular separation and sieving, and molecular flow manipulation in nanofluidic or microfluidic channels. The separation or sieving can be achieved when a suitable driving force is supplied to trapped molecules, when they migrate across many molecular traps and get separated because of the size-dependent trapping effect. Just before the migration through the thin regions, molecules are sieved by entropic trapping effect. After the molecules pass through the thin region, they relax back to their equilibrium shape quickly because of the existence of the thick regions. This process is repeated many times until the required separation is achieved. By controlling the driving force for the molecules, molecular trapping and manipulation can be achieved with the same structure.
In accordance with one embodiment of the invention, a new method was used to fabricate a nanofluidic channel having narrow constrictions (thin regions), spaced along the length of the channel, with the depth of the thin regions ranging between about 10 nm and about 500 nm, and having relatively thick regions between adjacent constrictions, of between about 0.5 micrometer and about 10 micrometer. Channels of these approximate dimensions may be referred to herein as nanofluidic channels, or simply as nanochannels. In accordance with this method, channels with variable depths were defined and etched in a silicon substrate, or wafer, using two-level photolithography. After a thermal oxidation process, mainly for electrical isolation, the top surface of the device was covered with a thin transparent plate. This technique permitted easy fabrication of very narrow gaps or constrictions without the need for e-beam lithography for the patterning of sub-micrometer features. This process was accomplished by the use of differential etching of two regions and the bonding of a capping layer.